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EP4177266A1 - Neutralisation d'anticorps humains anti-sars-cov-2 - Google Patents

Neutralisation d'anticorps humains anti-sars-cov-2 Download PDF

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EP4177266A1
EP4177266A1 EP21206787.0A EP21206787A EP4177266A1 EP 4177266 A1 EP4177266 A1 EP 4177266A1 EP 21206787 A EP21206787 A EP 21206787A EP 4177266 A1 EP4177266 A1 EP 4177266A1
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Prior art keywords
antibody
seq
sequence
sars
antigen binding
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Katholieke Universiteit Leuven
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Priority to PCT/EP2022/080965 priority patent/WO2023079137A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to antibodies which are particularly suitable in the treatment of beta and delta variant of COVID 19
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 spike protein consists of an S1 subunit that recognizes host cell receptors and an S2 subunit that promotes membrane fusion of virus and host cells.
  • the receptor binding domain Within the S1 subunit, the receptor binding domain (RBD) is responsible for interaction with receptor angiotensin-converting enzyme 2 (ACE2) on host cells to mediate viral entry. Consequently, SARS-CoV-2 spike protein is the major target of neutralizing antibodies (Ab) 2,3.
  • Antibodies can be elicited by natural infection and vaccination, or can be administered as recombinant monoclonal antibodies (mAbs) in a passive immunization strategy.
  • mAbs monoclonal antibodies
  • vaccines are essential tools to fight this pandemic
  • therapeutic mAbs play a crucial role as well, especially for (immune-compromised) individuals who may not generate a robust response to their vaccine, cannot be vaccinated, are at high risk for severe illness or are still awaiting their vaccine 4 .
  • REGN-COV2 i.e. REGN10933 + REGN10987
  • LY-CoV555 with/without LY-CoV016 from AbCellera/Eli Lilly and VIR-7831 from Vir Biotechnology/GlaxoSmithKline
  • C-P59 from Celltrion
  • SARS-CoV-2 variants beta, gamma and delta escape from some of the currently available therapeutic mAbs.
  • REGN10933 i.e.
  • Therapeutic monoclonal antibodies are an essential tool to combat COVID-19 morbidity and mortality, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • mAbs have already received emergency use authorization either in the United States or South Korea.
  • SARS-CoV-2 variants escape some of these therapeutic mAbs.
  • PBMCs from convalescent patients and B cell receptor cloning we identified six mAbs, classified in four different epitope groups, that potently neutralize live SARS-CoV-2 Wuhan, alpha, beta, gamma and delta infection in vitro.
  • antibodies 3E6 and 3B8 potently cure infection with SARS-CoV-2 Wuhan, beta and delta in an in vivo hamster model when administered therapeutically at 5 mg/kg.
  • antibody 3B8 still reduced infectious viral titers after therapeutic administration at only 0.2 mg/kg, highlighting its superior potency.
  • antibody 3B8 is an ultrapotent therapeutic antibody, with treatment at a dose of 1 mg/kg resulting in undetectable infectious viral titers. Even when administered at 0.2 mg/kg, viral replication was reduced, with median infectious viral titers of the treatment group being 67 times lower compared to the isotype control group. This further highlights the potency of antibody 3B8 in a therapeutic setting 2,34-38 . High potencies are crucial to decrease cost of goods, enable sustainable manufacturability and increase the number of doses produced annually, and may therefore also facilitate the availability of therapeutic mAbs to low- and middle-income countries.
  • K D values observed for our antibodies were consistently between 3- and 200-fold lower than values reported for marketed antibodies of Vir Biotechnology, Regeneron Pharmaceuticals or Eli Lilly 39-41 .
  • K D values were similar to values observed for our mAbs (data not shown).
  • antibodies 3B8 and 3E6 each performed equally well as monotherapy (when administered at 5 mg/kg) to treat SARS-CoV-2 infection beta and delta in hamsters when compared to the marketed REGN-COV2 antibody cocktail, which consists of two different antibodies 23 .
  • the specified binding moieties bind preferentially to a particular target protein and do not bind in a significant amount to other components present in a test sample.
  • Specific binding to a target protein under such conditions may require a binding moiety that is selected for its specificity for a particular target antigen.
  • a variety of assay formats may be used to select ligands that are specifically reactive with a particular protein, for example, solid-phase ELISA immunoassays, or immunoprecipitation.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background.
  • antibodies and antibody fragments of the invention preferentially bind to the RBD domain of the spike protein S1 subunit of Sars-Cov-2, whereby by “preferentially binding”, “preferentially recognizing” or “preferentially reacting with” is meant that the antibodies or antibody fragments show greater binding capacity for RBD domain of the spike protein S1 subunit of Sars-Cov-2 as compared to any other antigen.
  • the binding capacity of an antibody or antibody fragment to an antigen is reflective of its affinity and/or avidity for that antigen. Binding specificity can be expressed by association and dissociation constants as determined by ELISA or BIACORE.
  • antibody and “antibodies” are recognized in the art and refer to proteins also known as immunoglobulins that bind to antigens. It is to be understood that these terms encompass conventional vertebrate antibodies like IgA, IgD, IgE, IgG, IgM, IgT, IgX and IgY, composed of at least two heavy and two light chains, as well as antibodies only composed of two heavy chains (VHH antibodies, IgNAR, heavy-chain antibodies, single-domain antibodies or nanobodies), and single-chain antibodies. In the case of conventional antibodies, the antigen binding sites are contributed to by the variable domains of both the heavy and light chains (VH and VL).
  • variable domain refers to the part or domain of an antibody which is partially or fully responsible for antigen binding.
  • variable domains will be amino acid sequences that essentially consist of 4 framework regions (FRI to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), or any suitable fragment of such an amino acid sequence which usually contains at least some of the amino acid residues that form at least one of the CDR's.
  • Such variable domains and fragments are most preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold.
  • Each CDR may contribute to a greater or lesser extent to antigen binding by the antibody.
  • Single domain antibodies or heavy-chain antibodies can be found in camelids and sharks, and each of the antigen-binding sites of these antibodies is formed by a single heavy chain variable domain (VHH) only. Therefore, only three CDRs contribute to a greater or lesser extent to each antigen-binding site.
  • Single chain antibodies (scFv) are derived from conventional antibodies by translational fusion of the VH and VL domains, separated by a flexible linker, into a single antigen-binding domain. Framework sequences of an antibody may be altered without altering the antigenic specificity of the antibody, or in order to change the binding affinity of the antibody. Furthermore, conventional antibodies may switch classes or isotypes without substantially affecting antigen-binding characteristics.
  • CDR complementarity determining region
  • VH and VL variable regions of either H (heavy) or L (light) chains (abbreviated as VH and VL, respectively) and contains the amino acid sequences capable of specifically binding to antigenic targets.
  • CDR regions account for the specificity of the antibody for a particular antigenic determinant structure. Such regions are also referred to as “hypervariable regions.”
  • the CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains.
  • antibody fragment is meant a fragment of an antibody that largely retains antigen-binding capacity of the antibody from which it is derived. Therefore, an antibody fragment of the invention is capable of preferentially binding to RBD domain of the spike protein S1 subunit of Sars-Cov-2. Antigen-binding capacity is determined by the variable domain or domains, more particularly by 1, 2, 3, 4, 5 or 6 CDRs located in the VH and/or VL domains in the case of conventional and single-chain antibodies, and 1, 2 or 3 CDRs in the case of single-domain antibodies. Preferred antibody fragments of the invention therefore comprise antigen-binding sites comprising 1, 2, 3, 4, 5 or 6 CDRs.
  • Two or more CDRs may be physically separated from each other by connecting regions to provide a framework structure for the CDRs.
  • More preferred antibody fragments of the invention comprise antigen-binding sites comprising 1 or 2 variable domains. Examples of antibody fragments are well-known to the skilled person and include the monovalent antigen-binding fragments (Fab), bivalent F(ab')2 fragments, Fv fragments (e.g. single chain antibodies scFv), miniaturized antibodies, single-domain antibody fragments like nanobodies ( Nelson AL 2010, Antibody fragments: hope and hype. mAbs 2:77-83 ). Antibody fragments of the invention may be obtained by enzymatic or chemical proteolysis, or by recombinant DNA technology techniques well known to the skilled person.
  • Antibodies and antibody fragments of the invention may be further chemically conjugated, non-covalently bound, or translationally fused to other proteins.
  • Single chain antibodies scFv are an example of translational fusion between a VH and a VL domain.
  • Further examples are albumin-conjugated antibodies or antibody fragments, bivalent diabodies, and monospecific and bispecific tandem svFcs ( Nelson AL 2010, Antibody fragments: hope and hype. mAbs 2:77-83 ).
  • a single-chain antibody (also referred to as "scFv") can be prepared by linking a heavy chain V region and a light chain V region of an antibody (for a review of scFv see Pluckthun "The Pharmacology of Monoclonal Antibodies” Vol. 113, eds. Rosenburg and Moore, Springer Verlag, New York, pp.269-315 (1994 )).
  • Methods for preparing single-chain antibodies are known in the art (see, for example, US Patent Nos. 4,946,778 , 5,260,203 , 5,091,513 , and 5,455,030 ).
  • the heavy chain V region and the light chain V region are linked together via a linker, preferably, a polypeptide linker ( Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A, 1988, 85, 5879-5883 ).
  • the heavy chain V region and the light chain V region in a scFv may be derived from the same antibody, or from different antibodies.
  • an “Fv” fragment is the smallest antibody fragment, and contains a complete antigen recognition site and a binding site.
  • This region is a dimer (VH-VL dimer) wherein the variable regions of each of the heavy chain and light chain are strongly connected by a noncovalent bond.
  • the three CDRs of each of the variable regions interact with each other to form an antigen binding site on the surface of the VH-VL dimer.
  • a total of six CDRs from the heavy and light chains function together as an antibody's antigen-binding site.
  • variable region or a half Fv, which contains only three antigen-specific CDRs
  • a preferred antibody fragment of the present invention is an Fv fragment, but is not limited thereto.
  • Such an antibody fragment may be a polypeptide which comprises an antibody fragment of heavy or light chain CDRs which are conserved, and which can recognize and bind its antigen.
  • a Fab fragment also contains a light chain constant region and heavy chain constant region (CH1).
  • a Fab fragment also contains a light chain constant region and heavy chain constant region (CH1).
  • an antigen-binding fragment called a Fab fragment
  • Fab fragment containing the variable regions of a heavy chain and light chain, which serve as a single antigen-binding domain
  • an Fc the remaining portion
  • a Fab' fragment is different from a Fab fragment in that a Fab' fragment also has several residues derived from the carboxyl terminus of a heavy chain CH1 region, which contains one or more cysteine residues from the hinge region of an antibody.
  • a Fab' fragment is, however, structurally equivalent to Fab in that both are antigen-binding fragments which comprise the variable regions of a heavy chain and light chain, which serve as a single antigen-binding domain.
  • an antigen-binding fragment comprising the variable regions of a heavy chain and light chain which serve as a single antigen-binding domain, and which is equivalent to that obtained by papain digestion, is referred to as a "Fab-like antibody", even when it is not identical to an antibody fragment produced by protease digestion.
  • Fab'-SH is Fab' with one or more cysteine residues having free thiol groups in its constant region.
  • a F(ab') fragment is produced by cleaving the disulfide bond between the cysteine residues in the hinge region of F(ab')2.
  • Other chemically crosslinked antibody fragments are also known to those skilled in the art.
  • Pepsin digestion of an antibody yields two fragments; one is a F(ab')2 fragment which comprises two antigen-binding domains and can cross-react with antigens, and the other is the remaining fragment (referred to as pFc').
  • an antibody fragment equivalent to that obtained by pepsin digestion is referred to as a "F(ab')2-like antibody" when it comprises two antigen-binding domains and can cross-react with antigens.
  • Such antibody fragments can also be produced, for example, by genetic engineering.
  • Such antibody fragments can also be isolated, for example, from the antibody phage library described above.
  • F(ab')2-SH fragments can be recovered directly from hosts, such as E. coli, and then allowed to form F(ab')2 fragments by chemical crosslinking ( Carter et al., Bio/Technology 10:163-167 (1992 )).
  • Antibodies and antibody fragments of the invention may be further modified.
  • modifications include the addition of detectable enzymatic, fluorescent, luminescent, or radioactive marker groups or molecules that act in detection such as streptavidin.
  • Other examples include the chemical modification to alter the half-life of antibodies and antibody fragments, such as PEGylation.
  • effector moieties to antibodies and antibody fragments, such as toxins, radioisotopes, enzymes, cytokines, and antigens ( Nelson AL 2010, Antibody fragments: hope and hype. mAbs 2:77-83 ).
  • Antibodies or antibody fragments may be further modified into an antibody-derived scaffold or antibody-like scaffolds that largely retains antigen-binding capacity of the antibody or antibody fragments from which it is derived.
  • antibody-derived scaffolds or antibody-like scaffolds are domain antibodies (dAb) that selectively or preferentially bind the same epitope as a natural antibody, for instance dAb with fully human frameworks, for instance dAb fused to a human Fc domain or for instance nanobodies engineered in a molecule that has an IgG-like circulating half-life in humans or antibody fragments with retained antigen-binding capacity or domain antibody with active scaffolds for controlled and cell delivery.
  • dAb domain antibodies
  • Single domain antibodies can be engineered into antibody like fragments.
  • Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.
  • Single domain antibodies may be any of the art, or any future single domain antibodies.
  • Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine.
  • a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO9404678 for example.
  • variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins.
  • a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco or Elasmobranchii species for instance skates, rays (batoidea), and sharks (selachii).
  • Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention.
  • the single-chain polypeptide may be produced by various methods well known in the art such as genetic engineering technique and chemical synthesis.
  • the genetic engineering technique includes constructing a replicable cloning vector or expression vector, transforming the host cell with the vector, culturing the transformed host cell to express the nucleic acid in it, collecting and purifying the single-chain polypeptide.
  • the vector usually comprises the nucleic acid encoding one of the two single-chain polypeptides constituting the diabody-type bispecific antibody according to the present invention. In such case, the resulting two kinds of the vectors are preferably introduced into the same host cell.
  • the two kinds of nucleic acid encoding the different single-chain polypeptides from each other may be comprised in the same vector.
  • the term "replicable expression vector” or "expression vector” as used herein refers to a piece of DNA (usually double-stranded) that may comprise a fragment of a foreign DNA fragment inserted therein.
  • the foreign DNA is also defined as a "heterologous DNA", which cannot be found naturally in a host cell in interest.
  • the vector is used to carry or convey the foreign or heterologous DNA into an appropriate host cell. Once the vector is introduced into the host cell, it may be replicated independently from a chromosomal DNA of the host cell to produce copies of the vector and foreign DNA inserted therein.
  • the vector also comprises elements essential for translating the foreign DNA into a polypeptide so that the polypeptide molecules encoded by the foreign DNA will be synthesized very quickly.
  • the above vector means a DNA construct comprising an appropriate control sequence and DNA sequence that are operably linked together (i.e., linked together so that the foreign DNA can be expressed).
  • the control sequence includes a promoter for transcription, an optional operator sequence to regulate the transcription, a sequence encoding an appropriate mRNA ribosome-biding site, an enhancer, a polyadenylation sequence, and a sequence controlling the termination of transcription and translation.
  • the vector may further comprise various sequences known in the art, such as a restriction enzyme cleaving site, a marker gene (selection gene) such as a drug-resistant gene, a signal sequence, and a leader sequence. These sequences and elements may be optionally selected by those skilled in the art depending on the kinds of the foreign DNA and host cell, and conditions of culture medium.
  • the vector may be in any form such as a plasmid, phage particle, or just simply genomic insert. Once the appropriate host cell is transformed with the vector, the vector will be replicated or function independently from the genome of the host cell, or the vector will alternatively be integrated into the genome of the cell. Any cell known in the art may be used as the host cell, for example, there may be mentioned procaryotic cells such as including E. coli, eucaryotic cells such as mammalian cells such Chinese hamster ovary (CHO) cell and human cells, yeast, and insect cells.
  • procaryotic cells such as including E. coli
  • eucaryotic cells such as mammalian cells such Chinese hamster ovary (CHO) cell and human cells, yeast, and insect cells.
  • the single-chain polypeptide obtained by the expression in the host cell is usually secreted and collected from the culture medium, it may be also collected from cell lysate when it is directly expressed without a secretion signal.
  • the single-chain polypeptide may be released from the membrane with an appropriate surfactant such as Triton-X100.
  • Purification of the polypeptide may be carried out by any method known to those skilled in the art such as centrifugation, hydroxyapatite chromatography, gel electrophoresis, dialysis, separation on ion-exchange chromatography, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin-sepharose chromatography, anion-or cation-resin chromatography such as polyaspartic acid column, chromato-focusing, SDS-PAGE, precipitation with ammonium sulfate, and affinity chromatography.
  • the affinity chromatography which utilizes affinity with a peptide tag of the single-chain polypeptide, is one of the preferred purification techniques with a high efficiency.
  • the polypeptide is preferably purified after being solubilized and denatured.
  • the solubilization treatment may be carried out with the use of any agent known in the art, including alcohol such ethanol, a dissolving agent such as guanidine hydrochloride and urea.
  • the antibody-like fragments according to the present invention are produced by assembling the single-chain polypeptides, eventually on a scaffold, and separating and collecting the thus formed antibody-like fragments.
  • Assembling treatment brings the single-chain polypeptides back in an appropriate spatial arrangement in which a desired biological activity is shown. Thus, since this treatment brings the polypeptides or domains back into an assembling state, it may be considered “re-assembling.” It may be also called “re-constitution” or “refolding” in view of gaining the desired biological activity.
  • the assembling treatment may be carried out by any method known in the art preferably by gradually lowering the concentration of a denaturing agent such as guanidine hydrochloride in a solution comprising the single-chain polypeptide by means of dialysis.
  • a denaturing agent such as guanidine hydrochloride
  • an anti-coagulant or oxidizing agent may be optionally added in a reaction system in order to promote the oxidation.
  • the separation and collection of the formed antibody-like fragment may be done by any method known in the art as well.
  • VHHs are heavy chain variable domains derived from immunoglobulins naturally devoid of light chains such as those derived from Camelidae as described in WO9404678 (and referred to hereinafter as VHH domains or nanobodies).
  • VHH molecules are about 10 ⁇ smaller than IgG molecules. They are single polypeptides and very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of VHHs produces high yield, properly folded functional VHHs.
  • antibodies generated in Camelids will recognize epitopes other than those recognised by antibodies generated in vitro through the use of antibody libraries or via immunization of mammals other than Camelids or Elasmobranchii species ( WO 9749805 ).
  • anti-albumin VHH's may interact in a more efficient way with serum albumin which is known to be a carrier protein.
  • serum albumin which is known to be a carrier protein.
  • some of the epitopes of serum albumin may be inaccessible by bound proteins, peptides and small chemical compounds. Since VHH's are known to bind into 'unusual' or nonconventional epitopes such as cavities ( WO9749805 ), the affinity of such VHH's to circulating albumin may be increased.
  • the antibodies and antibody fragments of the invention are humanized.
  • Antibody fragments derived from an antibody of the invention can be fused to the Fc region of a human antibody, in order to obtain humanized antibodies and antibody fragments.
  • Humanized antibodies or antibody fragments can also be obtained by grafting of one or more CDRs or only their specificity-determining residues (SDRs), optionally together with one or more framework residues important for optimal CDR functionality, of a non-human antibody having the desired antigen-binding specificity, into framework polypeptide sequences of a human antibody or antibody fragment, or even into a universal humanized nanobody scaffold.
  • SDRs specificity-determining residues
  • humanized antibody as used herein means a human immunoglobulin (a recipient antibody) in which at least part of the residues of complementary-determining region (CDR) is replaced with residues derived from the CDR of a non-human animal antibody (a donor antibody) that has a desired specificity, affinity and capability, such as those of mouse, rat, and rabbit.
  • CDR complementary-determining region
  • a donor antibody a donor antibody
  • the residue(s) of a Fv framework (FR) in the human immunoglobulin is replaced with residue(s) of the corresponding non-human antibody.
  • the humanized antibody may further comprise a residue that is not found in the recipient antibody or the introduced CDR or framework.
  • the antibody fragments of the present invention can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display epitope-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an epitope-binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen bound or captured to a solid surface or bead.
  • Phages used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with antigen-binding antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Examples of phage display methods that can be used to make the antibodies or antibody fragments of the present invention include those disclosed in ( Kettleborough et al Eur. J. Immunol., 24:952-958 (1994 ); Burton et al., Advances in Immunology, 57: 191-280 (1994 ); Brinkman et al., J. Immunol. Methods, 182: 41-50 (1995 ); Ames et al., J. Immunol.
  • the regions of the phage encoding the fragments can be isolated and used to generate the epitope-binding fragments through expression in a chosen host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, using recombinant DNA technology.
  • a chosen host including mammalian cells, insect cells, plant cells, yeast, and bacteria.
  • techniques to recombinantly produce antigen-binding fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324 ; Better et al., Science, 240: 1041-1104 (1988 ); Mullinax et al., Biotechniques, 12(6):864-869 (1992 ); Sawai et al., AJRI, 34:2634 (1995 ).
  • Changes may be made to the residues that comprise the CDRs without interfering with the ability of the antibody to recognize and bind its cognate epitope. For example, changes that do not affect epitope recognition, yet increase the binding affinity of the antibody for the epitope may be made.
  • Several studies have surveyed the effects of introducing one or more amino acid changes at various positions in the sequence of an antibody, based on the knowledge of the primary antibody sequence, on its properties such as binding and level of expression ( Yang et al., J Mol Biol. 254[3]:392-403 (1995 ); Vaughan et al., Nat Biotechnol. 16[6]: 535-539 (1998 ); Rader et al., Proc Natl Acad Sci U.S.A.
  • the antibody of the invention is monoclonal.
  • the term "monoclonal antibody” is well recognized in the art and refers to an antibody or a homogenous population of antibodies that is derived from a single clone. Individual antibodies from a monoclonal antibody population are essentially identical, in that minor naturally occurring mutations may be present. Antibodies from a monoclonal antibody population show a homogenous binding specificity and affinity for a particular epitope.
  • percentage identity or percent sequence identity between two or more amino acid sequences or two or more nucleotide sequences refers to the ratio, expressed in %, of: the number of amino acids or nucleotides in an optimal alignment of the amino acid sequences or nucleotide sequences that are identical in both sequences (i.e. match), to the length of the alignment, i.e. the number of aligned positions, including gaps if any.
  • nucleic acid is intended to include DNA molecules and RNA molecules.
  • a nucleic acid can be single-stranded or double-stranded.
  • the nucleic acids of the invention are present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is "isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCI banding, column chromatography, agarose gel electrophoresis and others well known in the art. see, e.g., Sambrook, Tijssen and Ausubel.
  • nucleic acid sequences of the invention and other nucleic acids used to practice this invention may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed recombinantly. Any recombinant expression system can be used, including, in addition to bacterial, e.g., yeast, insect or mammalian systems. Alternatively, these nucleic acids can be chemically synthesized in vitro. Techniques for the manipulation of nucleic acids, such as, e.g., subcloning into expression vectors, labeling probes, sequencing, and hybridization are well described in the scientific and patent literature, see, e.g., Sambrook, Tijssen and Ausubel.
  • Nucleic acids can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, such as fluid or gel precipition reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.
  • analytical biochemical methods such as NMR, spect
  • nucleic acid compositions of the present invention while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures may be mutated, thereof in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired.
  • DNA sequences substantially homologous to or derived from such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence).
  • Table 1 overview of RBD-binding titers and neutralizing antibody titers in serum, and percentage RBD-positive B cells in PBMCs from 26 convalescent COVID-19 patients
  • Patient ID anti-RBD Ab ⁇ g/ml
  • Neutralizing Abs SNT50 titer
  • % RBD-positive of B cells K-COV19-901 182,16 1209 0,80% K-COV19-902 21,65 96 0,00% K-COV19-006 387,59 7022 0,00% K-COV19-007 5,98 / 0,00% K-COV19-009 7,57 8 0,04% K-COV19-010 15,58 74 0,03% K-COV19-019 26,07 771 0,05% K-COV19-021 30,80 506 0,19% K-COV19-028 25,75 493 0,00% K-COV19-034 48,82 810 0,02% K-COV19-041 8,66 201 0,09% K-COV19-04
  • Patient K-COV19-901 having both a high titer of neutralizing antibodies and a high number of RBD-specific B cells, was selected for the isolation of individual RBD-specific B cells via fluorescence-activated cell sorting (FACS), using biotinylated RBD (Wuhan isolate) combined with streptavidin-PE as bait ( Figure 7 ).
  • FACS fluorescence-activated cell sorting
  • biotinylated RBD Wood isolate
  • streptavidin-PE streptavidin-PE
  • a panel of 20 unique antibody sequences (labelled K-COV-901-X, further mentioned as “X”) was selected for in vitro production and subsequent characterization. All 20 antibodies retained RBD and trimeric spike antigen (Wuhan) binding in vitro when analyzed via ELISA or surface plasmon resonance (SPR) assays. In addition, antibodies showed picomolar affinities to the RBD antigen and trimeric spike antigen with equilibrium constants (K D values) ranging from 31 to 443 pM and 32 to 243 pM, respectively (Table 2).
  • VSV vesicular stomatitis virus
  • D614G SARS-CoV-2 spike protein
  • IC 50 half-maximal 50% inhibitory concentration
  • RBD antigens bearing the single mutations E484K, E484Q, N501Y and L452R present - alone or combined - in multiple SARS-CoV-2 variants (i.e. alpha, beta, gamma, delta and others), as well as RBD antigens bearing the double mutation E484Q, L452R or triple mutation K417N, E484K, N501Y, as present in SARS-CoV-2 variants kappa and beta respectively 12 , were used to assess binding and affinity of the 8 most potent antibodies via both ELISA and SPR.
  • RBD mutations N439K described as an antibody evasion mutant 13-15
  • RBD Y453F originating from Danish mink farms and a potential neutralization resistance mutation 16,17
  • Table 3 overview of single RBD mutants used in this study and their presence in different SARS-CoV-2 variants of concern, variants of interest, variants under monitoring or de-escalated variants.
  • SARS-CoV-2 variants were evaluated in a cytopathic effect neutralization test (CPENT) using live SARS-CoV-2 Wuhan, alpha, beta and gamma virus strains and Vero E6 cells, as well as in a plaque reduction neutralization test (PRNT) using live SARS-CoV-2 delta strain with the same cell line.
  • CPENT cytopathic effect neutralization test
  • PRNT plaque reduction neutralization test
  • Infection with SARS-CoV-2 Wuhan and alpha was best neutralized by antibodies 3B8 and 2B2 (IC 50 ⁇ 0.63 and ⁇ 0.16 nM respectively), while SARS-CoV-2 beta infection was best neutralized by antibodies 3B8 and 3E6 (IC 50 ⁇ 0.32 nM).
  • SARS-CoV-2 gamma IC 50 ⁇ 0.37 nM for 3B8 and 3E6
  • antibodies 2B11 and 1C11 lost their neutralizing activity against SARS-CoV-2 delta (IC50 > 67 nM).
  • antibody 3B8 was the most potent neutralizing mAb towards all variants, with IC 50 values ranging from 0.37 nM to 0.01 nM for neutralization of SARS-CoV-2 gamma and delta strain respectively ( Figure 2 and Table 5).
  • Table 5 Overview of in vitro neutralizing activity (IC 50 ) towards SARS-CoV-2 Wuhan, alpha, beta, gamma and delta for top 8 mAbs.
  • Neutralization - IC 50 (nM) Antibody CPE live virus assay ⁇ Wuhan CPE live virus assay ⁇ alpha CPE live virus assay ⁇ beta CPE live virus assay ⁇ gamma PRNT live virus assay ⁇ delta 1A10 3.94 0.85 3.21 1.38 1.39 1C1 2.31 0.55 2.56 2.53 1.05 1C11 1.53 0.9 5.23 3.92 ND 2B11 2.29 0.69 5.46 1.17 ND 2B2 0.63 0.16 3.02 2.31 3.97 2D6 3.34 0.82 2.94 1.28 0.98 3B8 0.25 0.1 0.11 0.37 0.01 3E6 2.19 0.85 0.32 0.19 1.08 Neutralizing activity (IC 50 ; in nM) was determined via CPE assay for SARS-CoV-2 Wuhan, alpha, beta and gamma and via PRNT assay for SARS-CoV-2 delta.
  • the eight selected neutralizing antibodies were also tested for their ability to cross-neutralize other human coronavirus strains using VSV pseudotypes expressing the spike protein from SARS-CoV, Middle East respiratory stress (MERS)-CoV or 229E-CoV on their surface.
  • MERS Middle East respiratory stress
  • 229E-CoV Middle East respiratory stress
  • the selected mAbs were classified in different categories based on their epitope specificity using an ELISA cross-competition assay where the antibodies were pairwise tested against one another. Two groups could be distinguished consisting of antibodies 1A10, 1C1 and 2D6 on the one hand and antibodies 2B2, 2B11 and 1C11 on the other hand. Antibodies 3B8 and 3E6 could not be classified in one of these groups ( Figure 3 ). This suggests that the 8 selected mAbs bind to 4 different regions of the RBD antigen.
  • Antibodies, 3B8, 3E6, 2B2 and 1C1 were selected for in vivo evaluation based on their in vitro neutralizing activity combined with their variability in epitope binding.
  • 30 hamsters were divided into 5 groups (6 animals/group) and infected intranasally with 50 ⁇ l of SARS-CoV-2 Wuhan virus suspension (containing approximately 2 ⁇ 10 6 tissue culture infective dose (TCID 50 )).
  • TCID 50 tissue culture infective dose
  • antibodies 3B8, 3E6, 2B2, 1C1 or an IgG1 isotype control were injected intraperitoneally at a dose of 5 mg/kg.
  • SARS-CoV-2 variants of concern beta and delta are more resistant to neutralization by vaccine-induced antibodies or currently available mAb treatments 6,18,19 , and given that SARS-CoV-2 delta is the most dominant variant worldwide 20,21 , we decided to evaluate in vivo treatment efficacy of our antibodies against both variants as well.
  • a study design similar to the first study was used, with 30 hamsters divided in 5 groups and intranasally infected with 50 ⁇ l of SARS-CoV-2 beta 22 or SARS-CoV-2 delta suspension (both containing approximately 1 ⁇ 10 4 TCID 50 ).
  • Viral RNA load in lung tissue was significantly reduced by treatment with antibody 3B8, 3E6 and REGN-CoV-2 compared to isotype control (p ⁇ 0.05 for 3B8 and 3E6; p ⁇ 0.01 for REGN-CoV-2), while the reduction observed for 2B2 was not statistically significant ( Figure 5b ).
  • No infectious viral titer in lung tissue (TCID 50 ) could be detected upon treatment with 3B8, 3E6 or REGN-CoV-2 (p ⁇ 0.01 compared to isotype control), while a viral titer was still detectable in four out of five animals treated with 2B2 ( Figure 5c ).
  • Antibodies 3B8 and 3E6 both completely abrogated infectious viral titers after infection with SARS-CoV-2 Wuhan, beta and delta when administered 24h post infection at 5mg/kg. As 3B8 was superior to 3E6 in in vitro neutralization experiments, this antibody was used in an in vivo dose-response experiment. Hamsters were intranasally infected with SARS-CoV-2 delta (1 ⁇ 10 4 TCID 50 ). At 24h post infection, isotype control (5 mg/kg) or antibody 3B8 (5 mg/kg; 1 mg/kg, 0.2 mg/kg or 0.04 mg/kg) was administered intraperitoneally. Animals were sacrificed at day 4 post infection.
  • EXAMPLE 6 DNA based vaccination.
  • 3B8 was delivered encoded in plasmid DNA (pDNA) in vivo, as an equimolar mixture of the 3B8 heavy and light chain.
  • Hamsters received an intramuscular pDNA injection in their right & left tibialis anterior and gastrocnemius muscle (pretreated with hyaluronidase), followed by electroporation. This allows for intramuscular production and systemic circulation of 3B8. A series of electrical pulses were thereby delivered at 120V, using a plate electrode.
  • Total pDNA amount delivered per hamster was 600 ⁇ g (75 ⁇ l pDNA, at 2 ⁇ g/ ⁇ l per muscle).
  • Intramuscular pDNA administration was performed either on day -10, day -7 or day -5 infection (6 animals per timepoint).
  • PBMCs peripheral blood mononuclear cells
  • PBMCs were resuspended in freezing medium consisting of 10% (v/v) dimethyl sulfoxide (DMSO; Merck) and 90% fetal bovine serum (FBS; GIBCO) for cryopreservation at -80°C or in liquid nitrogen for long term storage.
  • DMSO dimethyl sulfoxide
  • FBS fetal bovine serum
  • Human B cells were enriched from cryopreserved PBMCs using the EasySep TM Human B Cell Enrichment Kit (STEMCELL Technologies) according to the manufacturer's instructions. After purification, B cells were stained with Zombie Aqua TM Fixable Viability Kit (Biolegend) and blocked with FcR Blocking Reagent (Miltenyi Biotec) for 15 minutes on ice. Following the 15 minutes incubation, B cells were washed with FACS buffer (phosphate-buffered saline (PBS) + 2% FBS + 2 mM EDTA) and incubated with biotinylated SARS-CoV-2 RBD protein for 60 minutes at on ice.
  • FACS buffer phosphate-buffered saline (PBS) + 2% FBS + 2 mM EDTA
  • Histag labeled SARS-CoV-2 RBD (The Native Antigen Company) was biotinylated with the EZ-Link Sulfo-NHS-LC-Biotin kit (Thermofisher Scientific) according to the manufacturer's protocol, corresponding to 1-3 biotin groups per antibody molecule. After the 60 minutes incubation, B cells were washed with FACS buffer and stained with PerCP-cy5.5 anti-human CD19 antibody (Biolegend, 363016), FITC anti-human CD3 antibody (Biolegend, 300306) and PE streptavidin (Biolegend, 405203) for 25 minutes on ice.
  • the stained cells were washed twice with FACS buffer and single-cell sorted by BD Influx TM Cell Sorter (BD Biosciences). UltraComp eBeads TM Compensation Beads and tosylactivated M-280 Dynabeads (Invitrogen) coupled with SARS-CoV-2 RBD protein according to the manufacturer's instructions were used for compensation.
  • Selection of RBD-specific B cells was performed using the following gating strategy: lymphocytes were selected using FSC and SSC, followed by selection of single cells based on width versus area of FSC and SSC signals. Next, live cells were selected as Zombia Aqua negative cells.
  • B cells were selected as CD19+ CD3- cells and evaluated for RBD surface staining.
  • a fluorescence minus one (FMO) control was included ( figure 7 ).
  • Individual B cells were sorted into 96-wells PCR plates (Bioké) containing 2 ⁇ L lysis buffer (0.2% (v/v) Triton X-100 + 2U/ ⁇ L RNaseOUT TM Recombinant Ribonuclease (Invitrogen) in UltraPure TM DNAse/RNase free distilled water (Invitrogen)) per well.
  • the plates were sealed with a Microseal ® 'F' Film (BioRad) and immediately frozen on dry ice before storage at ⁇ 80°C for further use.
  • Transcripts of lysed single B cells were denatured and hybridized with a mix of 1 ⁇ L 10 mM each nucleotide dNTP-Mix (Invitrogen) and 1 ⁇ L 10 ⁇ M oligo-dT primerat 72°C for 3 minutes. Subsequently, cDNA synthesis and pre-amplification was performed according to Picelli et al 43 . cDNA was stored at ⁇ 20°C. cDNA was purified with Agencourt AMPure XP beads (Beckman Coulter) according to the manufacturer's protocol and quantified on the BioAnalyzer 2100 instrument (Agilent) and on a Qubit TM fluorometer (Thermofisher Scientific).
  • the sequences encoding the immunoglobulin (Ig) heavy- and light-chain variable regions (V H and V L ) of IgM and IgG were amplified using reverse primers described by Ozawa et al 44 . and forward primer described by Picelli et al 43 .
  • the PCR reaction was performed with KAPA HiFi HotStart ReadyMix (Roche) at 95°C for 3 min, followed by 30 cycles at 98°C for 20 seconds, 60°C for 45 seconds and 72°C for 60 seconds and terminated at 72°C for 5 minutes. All PCR products were checked on a 1% agarose gel and purified using the Agencourt AMPure XP beads. Concentrations were measured on a Qubit TM fluorometer for Sanger sequencing.
  • the Ig V H and V L sequences were cloned into a pcDNA3.4 expression vector containing human IgG1 constant regions.
  • the plasmid DNA (pDNA) was amplified by transformation in DH5a E. coli and subsequent purification using the NucleoBond Xtra Maxi EF kit (Machery - Nagel) according to the manufacturer's instructions. Plasmid purity and integrity was checked on a 1% agarose gel.
  • Recombinant antibodies were transiently transfected and produced in vitro in 293F Freestyle suspension cells (Thermofisher Scientific). Briefly, equal amounts of heavy- and light chain pDNA were mixed with X-tremeGENE HP DNA Transfection Reagent (Roche) in Freestyle TM 293 Expression Medium (Thermofisher Scientific) according to the manufacturer's protocol and incubated for at least 15 minutes to allow the pDNA to enter the liposomes. The transfection reagent mixture was added in a dropwise manner to 293F Freestyle suspension cells.
  • the cells were cultured in T175 flasks (Sarstedt, Germany) on an orbital shaker (Thermofischer Scientific) at 135 rpm and 8% CO 2 in a 37°C humidified incubator for 5 days. At day 5, supernatant containing the recombinant antibodies was collected by centrifugation (1,400 ⁇ g for 10 minutes at room temperature), filtered through a 0.2 ⁇ m filter unit (VWR) and stored at - 20°C.
  • VWR 0.2 ⁇ m filter unit
  • the recombinant antibodies were eluted from the column with 100 mM sodium acetate, pH 3.5 and the collected fractions were immediately neutralized with 1M Tris, pH 9. The fractions were pooled and dialyzed against sterile-filtered PBS. The ⁇ KTAprime plus system and all glasswork was treated with 30% hydrogen peroxide (VWR) to reduce the presence of endotoxin.
  • VWR hydrogen peroxide
  • Purified recombinant antibodies were assessed by SDS-PAGE under nonreducing conditions using the Amersham Phastsystem TM (GE Healthcare) according to the manufacturer's instructions. Endotoxin levels were measured on Endosafe ® -PTS TM (Charles River) according to the manufacturer's instructions.
  • polystyrene 96-well flat-bottom microtiter plates (Corning costar) were coated with 100 ⁇ L/well of 2 ⁇ g/mL antigen diluted in PBS and incubated overnight at 4°C. The next day, plates were blocked with 200 ⁇ L/well blocking buffer containing 1% (w/v) Bovine Serum Albumin (BSA, Sigma Aldrich) in PBS for 2 hours at room temperature. The plates were then washed six times with wash buffer (0.002% (v/v) Tween-80 (Sigma Aldrich) in PBS). After washing, 100 ⁇ L/well of the samples, calibrators and/or controls were added.
  • BSA Bovine Serum Albumin
  • an anti-SARS-CoV-2 RBD mAb 40150-D004, Sino Biological
  • anti-SARS-CoV-2 nucleocapsid antibody MFS2563841, MyBioSource
  • All samples and controls were diluted in PBS + 0.1% (w/v) BSA + 0.002% (v/v) Tween 80 with or without 1,86 g/L EDTA (PTA(E) buffer). After incubation, the plates were washed six times and incubated with 100 ⁇ L/well horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Fc-specific, Sigma Aldrich) diluted 1/5000 in PTA buffer.
  • HRP horseradish peroxidase
  • Polystyrene 96-well flat-bottom microtiter plates were coated with 4 ⁇ g/mL purified recombinant capture antibody in PBS (100 ⁇ L/well) and incubated at 4°C. After an overnight incubation, plates were blocked with 200 ⁇ L/well blocking buffer for 2 hours at room temperature. The plates were washed six times with washing buffer and 10 ng/mL SARS-CoV-2 RBD protein diluted in PTA buffer (100 ⁇ L/well) was added to the plates for a 2-hour incubation at room temperature.
  • SPR Surface Plasmon Resonance
  • Monoclonal Abs were then captured between 30 and 100 RU. Increasing concentrations of analyte were sequentially injected in one single cycle at a flow rate of 30 ⁇ l/min. The dissociation was monitored for 30 min. The chip was finally regenerated with 3M MgCl2 before a new mAb was captured. A reference flow was used as a control for non-specific binding and refractive index changes. Several buffer blanks were used for double referencing. Binding affinities (KD) were derived after fitting the experimental data to the 1:1 binding model in the Biacore T200 Evaluation Software 3.1 using the single cycle kinetic procedure. Each interaction was repeated a least three times.
  • SARS2, SARS1, MERS, 229E Production of S-pseudotyped virus and serum neutralization test (SARS2, SARS1, MERS, 229E)
  • CPE Cell-based cytopathic (CPE) assay and CPE-based virus neutralization test (CPENT) (SARS-CoV-2 Wuhan, alpha, beta, gamma)
  • the titers of the virus stocks (TCID50) and 50% neutralizing titers (log 10 CPENT 50 ) were determined using cytopathic effect (CPE)-based cell assays and CPE-based virus neutralization tests (CPENT), respectively, on VeroE6 cells with some modifications. Shortly, 10 4 VeroE6 cells/well were seeded in 96-well plates one day prior to the titration and inoculated with 10-fold serial dilutions of virus solutions and cultured for 3 days at 37°C. Later, assays first visually scored for CPE and then stained with MTS/Phenazine methosulphate (PMS; Sigma- Aldrich) solution for 1.5 h at 37°C in the dark.
  • CPE cytopathic effect
  • CPENT CPE-based virus neutralization tests
  • Post MTS/PMS staining absorbance was measured at 498 nm for each well. All assays were performed in six replicates and TCID 50/ ml was determined using Reed and Muench method 46 . CPENT was measured in a similar way as the CPE assays for viral titration with some modifications. In brief, serial dilutions of antibodies were mixed separately with live SARS-CoV-2 Wuhan, alpha, beta and gamma virus strains, incubated at 37°C for 1h, and added to the monolayer of Vero E6 cells. All distinct antibodies were assayed in triplicate in serial dilutions.
  • SARS-CoV-2 plaque reduction neutralization test PRNT
  • SARS-CoV-2 delta SARS-CoV-2 delta
  • the PRNT experiments were performed with a SARS-CoV-2 isolate from the B.1.617.2 lineage. This virus was isolated from oropharyngeal swabs taken from a patient in Belgium (EPI_ISL_2425097; 2021-04-20). Virus stocks were prepared by 2x passaging on Vero E6 cells.
  • Dose-dependent neutralization of distinct antibodies was assessed by mixing the antibodies at different concentrations, with 100 PFU SARS-CoV-2 in DMEM supplemented with 2% FBS and incubating the mixture at 37°C for 1h. Antibody-virus complexes were then added to VeroE6 cell monolayers in 12-well plates and incubated at 37°C for 1h. Subsequently, the inoculum mixture was replaced with 0.8% (w/v) methylcellulose in DMEM supplemented with 2% FBS. After 3 days incubation at 37°C, the overlays were removed, the cells were fixed with 3.7% PFA, and stained with 0.5% crystal violet.
  • Half-maximum neutralization titers PRNT50 were defined as the antibody concentration that resulted in a plaque reduction of 50%.
  • BetaCov/Belgium/GHB-03021/2020 (EPI ISL 109 407976
  • a close relation with the prototypic Wuhan-Hu-1 2019-nCoV (GenBank accession 112 number MN908947.3) strain was confirmed by phylogenetic analysis. Infectious virus was isolated by serial passaging on Huh7 and Vero E6 cells 47 ; passage 6 virus was used for the study described here.
  • Beta variant B.1.351 (hCoV-19/Belgium/rega-1920/2021; EPI_ISL_896474, 2021-01-11) was isolated from nasopharyngeal swabs taken from a traveler returning to Belgium in January 2021 who became a patient with respiratory symptoms.
  • the Delta variant, B.1.617.2 (hCoV-19/Belgium/rega-7214/2021; EPI_ISL_2425097; 2021-04-20) was isolated from nasopharyngeal swabs in April 2021 in Belgium through active surveillance. Both strains were subjected to sequencing on a MinION platform (Oxford pore) directly from the nasopharyngeal swabs.
  • Infectious virus was isolated by passaging on VeroE6 cells 22 ; passage 2 was used for the study described here. Live virus-related work was conducted in the high-containment A3 and BSL3+ facilities of the KU Leuven Rega Institute (3CAPS) under licenses AMV 30112018 SBB 219 2018 0892 and AMV 23102017 SBB 219 20170589 according to institutional guidelines.
  • 3CAPS KU Leuven Rega Institute
  • Vero E6 cells African green monkey kidney, ATCC CRL-1586 were cultured in minimal essential medium (MEM, Gibco) supplemented with 10% fetal bovine serum (Integro), 1% non-essential amino acids (NEAA, Gibco), 1% L- glutamine (Gibco) and 1% bicarbonate (Gibco). End-point titrations on Vero E6 cells were performed with medium containing 2% fetal bovine serum instead of 10%.
  • Huh7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Integro), 1% bicarbonate (Gibco), 2% HEPES buffer (Gibco). Both cells were kept in a humidified 5% CO 2 incubator at 37°C.
  • the hamster infection model of SARS-CoV-2 has been described before 47,48 .
  • Female Syrian hamsters (Mesocricetus auratus) were purchased from Janvier Laboratories and kept per two in individually ventilated isolator cages (IsoCage N Bio-containment System, Tecniplast) at 21°C, 55% humidity and 12:12 day/night cycles. Housing conditions and experimental procedures were approved by the ethics committee of animal experimentation of KU Leuven (license P065-2020).
  • Lungs were collected and viral RNA and infectious virus were quantified by RT-qPCR and end-point virus titration, respectively. Blood samples were collected at end-point for pharmacokinetic analysis. No randomization methods were used and confounders were not controlled, though all caretakers and technicians were blinded to group allocation in the animal facility and to sample numbers for analysis (qPCR, titration, and histology).
  • RT-qPCR was performed on a LightCycler96 platform (Roche) using the iTaq Universal Probes One-Step RT-qPCR kit (BioRad) with N2 primers and probes targeting the nucleocapsid 48 .
  • Standards of SARS-CoV-2 cDNA (IDT) were used to express viral genome copies per mg tissue 47 .
  • Lung tissues were homogenized using bead disruption (Precellys) in 350 ⁇ L minimal essential medium and centrifuged (10,000 rpm, 5min, 4°C) to pellet the cell debris.
  • Precellys bead disruption
  • endpoint titrations were performed on confluent Vero E6 cells in 96- well plates.
  • Viral titers were calculated by the Reed and Muench method 46 using the Lindenbach calculator and were expressed as 50% tissue culture infectious dose (TCID 50 ) per mg tissue.

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